Dysregulated Signaling at Postsynaptic Density: A Systematic Review and Translational Appraisal for the Pathophysiology, Clinics, and Antipsychotics’ Treatment of Schizophrenia

Emerging evidence from genomics, post-mortem, and preclinical studies point to a potential dysregulation of molecular signaling at postsynaptic density (PSD) in schizophrenia pathophysiology. The PSD that identifies the archetypal asymmetric synapse is a structure of approximately 300 nm in diameter, localized behind the neuronal membrane in the glutamatergic synapse, and constituted by more than 1000 proteins, including receptors, adaptors, kinases, and scaffold proteins. Furthermore, using FASS (fluorescence-activated synaptosome sorting) techniques, glutamatergic synaptosomes were isolated at around 70 nm, where the receptors anchored to the PSD proteins can diffuse laterally along the PSD and were stabilized by scaffold proteins in nanodomains of 50–80 nm at a distance of 20–40 nm creating “nanocolumns” within the synaptic button. In this context, PSD was envisioned as a multimodal hub integrating multiple signaling-related intracellular functions. Dysfunctions of glutamate signaling have been postulated in schizophrenia, starting from the glutamate receptor’s interaction with scaffolding proteins involved in the N-methyl-D-aspartate receptor (NMDAR). Despite the emerging role of PSD proteins in behavioral disorders, there is currently no systematic review that integrates preclinical and clinical findings addressing dysregulated PSD signaling and translational implications for antipsychotic treatment in the aberrant postsynaptic function context. Here we reviewed a critical appraisal of the role of dysregulated PSD proteins signaling in the pathophysiology of schizophrenia, discussing how antipsychotics may affect PSD structures and synaptic plasticity in brain regions relevant to psychosis.


Introduction: The PSD Landscape, Receptors, Adaptors, and Scaffolding Proteins Linking to Schizophrenia
Among the major components of the glutamatergic synaptic complex, the postsynaptic density (PSD) is a finely specialized structure that lies beneath the postsynaptic membrane [1]. Several lines of evidence converge in envisioning PSD as a molecular switchboard that dispatches with spatial and temporal differentiation multimodal intracellular signaling from receptors lying on the postsynaptic surface, including N-methyl-Daspartate receptor (NMDAR), metabotropic glutamate receptor (mGluR) type I, α-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor (AMPAR) kainate receptor, to PSD proteins acting as effectors/scaffolds, including postsynaptic density protein 95 (PSD-95), disrupted in schizophrenia 1 (DISC1), stargazin, Homer, SH3, and multiple ankyrin repeat domains (Shank), and finally to vesicular compartments with both Ca 2+ -dependent 1.
How is PSD involvement coherent within the present framework of aberrant dopamineglutamate interaction in schizophrenia? 2.
What is the contribution of each PSD protein associated with the pathophysiology of schizophrenia? 3.
How might innovative methodology and discovery contribute to a non-canonical conceptualization of PSD's role in schizophrenia? 4.
Finally, how does antipsychotic treatment impact PSD proteins, and how might it be instrumental in developing new therapeutic strategies for schizophrenia?

Materials and Methods
The present systematic review aims to provide an updated overview of the available evidence on the implication of PSD proteins in dysregulated signaling transduction and their potential implications in antipsychotic treatment in schizophrenia. The search and selection process were conducted according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines [17] in order to identify eligible clinical and preclinical studies investigating the signaling dysregulations at the PSD in schizophrenia. Therefore, the following searches were conducted in EMBASE, Scopus, and PubMed on 1 August 2022 (the last interrogation was conducted in 28 October 2022): (psd) AND (schizophrenia) + "psd schizophrenia" OR (psd AND ("schizophrenia"/exp OR schizophrenia)); ((schizophrenia) AND (psd)) AND (antipsychotic*) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "antipsychotic*"; ((schizophrenia) AND (psd)) AND (clozapine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "clozapine"; ((schizophrenia) AND (psd)) AND (aripiprazole) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "aripiprazole"; ((schizophrenia) AND (psd)) AND (brexpiprazole) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "brexpiprazole"; ((schizophrenia) AND (psd)) AND (cariprazine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "cariprazine"; ((schizophrenia) AND (psd)) AND (risperidone) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "risperidone"; ((schizophrenia) AND (psd)) AND (haloperidol) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "haloperidol"; ((schizophre-Cells 2023, 12, 574 3 of 61 nia) AND (psd)) AND (olanzapine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "olanzapine"; ((schizophrenia) AND (psd)) AND (chlorpromazine) + ("schizophrenia"/ exp OR schizophrenia) AND psd AND "chlorpromazine"; ((schizophrenia) AND (psd)) AND (fluphenazine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "fluphenazine"; ((schizophrenia) AND (psd)) AND (lurasidone) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "lurasidone"; ((schizophrenia) AND (psd)) AND (xanomeline) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "xanomeline"; ((schizophrenia) AND (psd)) AND (quetiapine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "quetiapine"; ((schizophrenia) AND (psd)) AND (iloperidone) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "iloperidone"; ((schizophrenia) AND (psd)) AND (sulpiride) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "sulpiride"; ((schizophrenia) AND (psd)) AND (amisulpride) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "amisulpride"; ((schizophrenia) AND (psd)) AND (asenapine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "asenapine"; ((schizophrenia) AND (psd)) AND (bromperidol) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "bromperidol"; ((schizophrenia) AND (psd)) AND (clotiapine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "clotiapine"; ((schizophrenia) AND (psd)) AND (loxapine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "loxapine"; ((schizophrenia) AND (psd)) AND (paliperidone) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "paliperidone"; ((schizophrenia) AND (psd)) AND (perphenazine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "perphenazine"; ((schizophrenia) AND (psd)) AND (pimozide) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "pimozide"; ((schizophrenia) AND (psd)) AND (promazine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "promazine"; ((schizophrenia) AND (psd)) AND (thioridazine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "thioridazine"; ((schizophrenia) AND (psd)) AND (tiapride) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "tiapride"; ((schizophrenia) AND (psd)) AND (trifluoperazine) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "trifluoperazine"; ((schizophrenia) AND (psd)) AND (ziprasidone) + ("schizophrenia"/exp OR schizophrenia) AND psd AND "ziprasidone". The PRISMA flow diagram is reported in Figure 1. We deemed eligible English-written articles, published in peerreviewed journals, exploring the involvement of dysregulated signaling of PSD proteins in schizophrenia and their implication in antipsychotic treatment. No time constraints were applied, and original clinical and preclinical research studies and reviews were included. Conference abstracts and commentaries were excluded. The search returned a total of 1300 articles. One hundred and twenty-one articles were included in the qualitative synthesis and divided into topics of interest. The selection of articles from the primary search was conducted independently by two authors, and, in case of inconsistent evaluation, a third author's opinion was requested. Furthermore, in addition to the primary PRISMA search and after having evaluated the abstract and full text of each article of the final selection, a manual and ad hoc search was performed with a focus on PSD proteins with the highest citation for putative association/involvement in schizophrenia. This additional search was necessary when the primary mechanisms or functions of PSD proteins needed to be precise in light of the other molecular mechanisms of schizophrenia pathophysiology (i.e., dopamine-glutamate interaction). Although this may introduce a search bias, we should acknowledge it; nevertheless, we believe that for the present topic, mainly a translational review linking preclinical and clinical data, which is different from a more oriented clinical systematic review (i.e., interventional or observational ones), the overall strategy has allowed us to deepen the mechanistic aspects of PSD involvement in schizophrenia. We registered the review protocol on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY2022110129). In addition, after reading the entire text of the selected article, other cross-references were included. the present topic, mainly a translational review linking preclinical and clinical data, which is different from a more oriented clinical systematic review (i.e., interventional or observational ones), the overall strategy has allowed us to deepen the mechanistic aspects of PSD involvement in schizophrenia. We registered the review protocol on the International Platform of Registered Systematic Review and Meta-analysis Protocols (INPLASY2022110129). In addition, after reading the entire text of the selected article, other cross-references were included.

The Spatial and Temporal Distribution of PSD
Recent evidence has highlighted the relevance of PSD proteins, such as PSD-95, Homer, Shank, and DISC1, in the pathophysiology of major psychiatric disorders, including schizophrenia and autism spectrum disorder (ASD). A highly conserved set of ~1000 PSD proteins is shared among vertebrates, and differences in the proteome indicate how species have evolved distinct synapse types and functions [18]. The PSD has is a thickness of approximately 300-500 Angstrom length detectable with electron microscopy at the postsynaptic membrane of glutamatergic synapses. Applying two different types of targeted mass spectrometry-based assays in mouse cortical brain tissue, 50 PSD proteins have been detected with a parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards, whereas 2100 proteins were identified based on a pre-determined spectral library in PSD fractions [19]. Two major layers have been identified with electron microscopy in the overall structure of the PSD that is from the external part of the membrane surface are: (1) the "core" immediately beneath the neuronal membrane and (2) the "pallium" based on the typology of proteins therein localized and composed of scaffold proteins, receptors, adaptors, and inhibitory proteins. It is considered a structural and functional intersection where several neurotransmitter systems, such as glutamatergic, dopaminergic, and serotoninergic, converge and is

The Spatial and Temporal Distribution of PSD
Recent evidence has highlighted the relevance of PSD proteins, such as PSD-95, Homer, Shank, and DISC1, in the pathophysiology of major psychiatric disorders, including schizophrenia and autism spectrum disorder (ASD). A highly conserved set of 1000 PSD proteins is shared among vertebrates, and differences in the proteome indicate how species have evolved distinct synapse types and functions [18]. The PSD is a thickness of approximately 300-500 Angstrom length detectable with electron microscopy at the postsynaptic membrane of glutamatergic synapses. Applying two different types of targeted mass spectrometry-based assays in mouse cortical brain tissue, 50 PSD proteins have been detected with a parallel reaction monitoring (PRM) assay using heavy-labeled, synthetic internal peptide standards, whereas 2100 proteins were identified based on a pre-determined spectral library in PSD fractions [19]. Two major layers have been identified with electron microscopy in the overall structure of the PSD that is from the external part of the membrane surface are: (1) the "core" immediately beneath the neuronal membrane and (2) the "pallium" based on the typology of proteins therein localized and composed of scaffold proteins, receptors, adaptors, and inhibitory proteins. It is considered a structural and functional intersection where several neurotransmitter systems, such as glutamatergic, dopaminergic, and serotoninergic, converge and is believed to have a potentially relevant implication in the pathophysiology of psychosis [20]. With regard to glutamatergic neurotransmission, NMDAR, which is considered the core of the structure in concert with AMPAR and mGluR type I (mGluR 1 and mGluR 5) [21] plays a crucial role in the PSD complex. The most abundant membrane-associated guanylate kinase family (MAGUK) of mammalian PSD is postsynaptic density protein 95 (PSD-95), discs large 4 (DLG4), and synapse-associated protein 90 (SAP90). Others include SAP97 (DLG1), PSD-93 (DLG2, Chapsyn-110), SAP102 (DLG3), guanylate kinase-associated protein (GKAP), also called SAPAP (SAP90/PSD-95-associated protein), SH3 and multiple ankyrin repeat domains protein (Shank) also known as ProSAP (proline-rich synapse-associated protein), Homer otherwise called Vesl, Cupidin, and PSD-Zip45. Thus, in the dense matrix of PSD, the most represented scaffolding and adaptor proteins associated with schizophrenia pathophysiology, are PSD-95, Homer, and Shank. These interacting proteins, which have the role of linking membrane surface receptors through their structure and function, modulate the intracellular signaling mechanism [22]. The PSD has emerged in the last few years as a relevant synaptic element involved in shaping the dendritic spines and regulating multiple cell transduction signaling, whose dysregulation has been linked to synaptic aberration in neurodevelopmental disorders [11,23,24]. In addition, multiple lines of evidence suggest that antipsychotic treatments, including novel interventions in refractory psychosis [12], impact PSD gene expression [9]. Electron microscopy studies identified a three-dimensional laminate network of scaffolding proteins that organizes signal detection and modulation by transducing a postsynaptic signal. This architecture is derived from the binding properties and characteristics of each protein of PSD [25]. PSD-95 is located in a layer just below the postsynaptic membrane, in a position favoring interaction between glutamate receptors, ion channels, signaling proteins, and cell adhesion molecules (CAMs) and clusters them on the postsynaptic membrane. Scaffold molecules not anchored to the postsynaptic membrane, such as Shank, guanylate-kinase associated proteins (GKAP), and Homer, fill a deeper layer within the PSD and favor the connection of synaptic membrane surface to cytoskeletal components (such as α-actinin, cortactin, and actin), regulating the size and shape of dendritic spines [26]. Centrally placed within the PSD lies the Shank family of proteins and their binding partners that form large multi-protein complexes and are considered the main scaffold within the PSD cytomatrix [27]. Moreover, PSD mainly consists of cytoskeletal proteins and calcium/calmodulin-dependent protein kinase II (CaMKII), while traditional scaffold proteins, such as PSD-95 and Shank1, are less represented components [22]. The temporal distribution of synaptic proteins is considered crucial for starting, modulating, and ending the intracellular signaling originating at the receptor level and conducted by adaptors, scaffolding, and effector proteins. In this regard, recently, coupling the halo-binding site/ligand technology with synaptosome mapping pipeline technology (SYNMAP) has allowed linking the HaloTag ligand to fluorophores, enabling irreversible labeling and visualization of tagged proteins [28]. These approaches could deepen the interaction of multiple PSD proteins, their spatial and temporal distribution in dendritic spines, and their mutual interplay.

Glutamate Receptors: NMDAR and Beyond
At the level of excitatory synapses, it has been shown that approximately 2000-3000 glutamate molecules are released from the synaptic vesicle, whereas only ∼50-100 ionotropic glutamate receptors are expressed by a single PSD. Therefore, more than 90% of glutamate molecules will escape the synaptic cleft even when the maximal binding capacity (B max ) of the ionotropic glutamate is elevated, as the B max is the total number of receptors detectable in brain tissue by a binding assay of reference radioligands, for example [ 3 H]MK801 for the NMDAR, (S)-[ 3 H]-5-fluorowillardiine for the AMPAR, or [ 3 H]kainic for kainite [29]. Furthermore, it has been shown that within a millisecond, the concentration of glutamate at the release site decreases by an order of magnitude. In this highly dynamic scenario, it is conceivable that the concentration, localization, diffusion, and activation/inactivation of the receptors must be precisely regulated, and PSD proteins are crucial to this mechanism [30][31][32][33]. Several types of glutamate receptors are grouped within the PSD, including metabotropic glutamate receptors, which mediate transmembrane signal transduction through interactions with G-proteins, along with ionotropic glutamate receptors, such as NMDAR, AMPAR, and kainate receptors. Among these, the two principal classes, NMDAR and AMPAR, are highly enriched in the PSD and are crucial for translating glutamate into electrical depolarization at the postsynaptic level. The binding of these receptors to PSD is mediated by physical interaction with a large number of supporting molecules, including PSD-95, guanylate kinase-associated protein (GKAP), glutamate receptor-interacting protein (GRIP), proteins interacting with C kinase-1 (PICK1), Shank, and Homer. Preclinical studies showed that the long-term potentiation (LTP) induction threshold decreased with The ProSAP/Shank family is constituted of PSD scaffolding proteins that are implicated in propagating and modulating glutamate neurotransmission [38]. Shank genes derive from the same orthologous genes, with slight differences in humans and animals, thus providing an optimal candidate for translational research in neuropsychiatric disorders [39]. Shank proteins (i.e., Shank1, Shank2, and Shank3) are coded by three different genes, each of them having multiple splice variants producing different Shank proteins [40]. The distinct Shank isoforms differ in their expression pattern. Shank1 is expressed in the rat brain [40,41] with high abundance in the cortex, hippocampus, and amygdala, with lower expression in the thalamus and substantia nigra and negligible levels in the cerebellum, caudate nucleus, corpus callosum and subthalamic nucleus [41]. Shank2 is strongly expressed in the cortex, hippocampus, Purkinje cells, olfactory bulb, and central grey cells [42]; Shank3, finally, is limitedly expressed in the rat brain [40]. At the PSD, these proteins are capable of interacting with structural elements as well as ligand-gated ion channels, cell adhesion molecules, such as neuroligins, and proteins regulating the dynamic assembly of filamentous actin (F-actin) [43]. Shank3-dependent molecular, structural, and functional changes of excitatory synapses have been characterized from preclinical in vivo studies in a complete KO mouse model of the autism-associated Shank3 gene, underlying pathophysiological mechanisms [44]. This model demonstrated that Shank3 deficiency might impair the mGluR5-Homer interaction, resulting in cortico-striatal circuit abnormalities that underlie learning deficits and ASD-like behavior, providing a novel potential therapeutic approach for Shank3-associated functional and behavioral defects in neuropsychiatric disorders [44,45].

Shank Connectome and Signalosome
Shank proteins may cross-link Homer and PSD-95 at the PSD and participate in NM-DAR, mGluR, and AMPAR downstream signaling [46]. Moreover, Shank proteins promote spine formation, as well as maturation and enlargement of dendritic spines [47]. Through their multiple interactions, Shank proteins enable the formation of a polymeric network complex, which requires the assembly of Homer tetramers and Shank multimers, forming a structure that has been proposed to serve as a functional platform for other PSD proteins [38]. Multiple lines of evidence showed that ASD-associated mutations in Shank3 may impair AMPAR and NMDAR signaling and may alter neurexin-neuroligin-mediated signaling in cultures of rat hippocampal neurons [48]. These findings may suggest that Shank protein aberrations could contribute to cognitive symptoms in intellectual disability and could also be implicated in schizophrenia [48]. Shank abnormalities have been described to impair glutamate-mediated neurotransmission and the morphology of dendritic spines. Indeed, Shank proteins appear to be critically involved in regulating the morphology, architecture, and function of dendritic spines and in modulating glutamatergic signaling Cells 2023, 12, 574 7 of 61 relevant to several neurodevelopmental disorders [49], such as schizophrenia [50]. Taken together, this evidence demonstrates a critical role for Shank in the normal development of neuronal connectivity and establishes causality between a disruption in the Shank genes and the biological basis of neuropsychiatric disorders [51].

PSD
The ErbB4 gene has no effect on either inhibitory synapses in interneurons or excitatory synapses in pyramidal neurons. NRG1 and ErbB4 signaling are necessary for the postnatal cortical GABA network.
Li et al., 2020 [77] Preclinical study Semi-quantitativeRT-PCR Immunofluorescence Mice Neuronal ErbB-4 is most prevalent in GABAergic interneurons. The fraction of ErbB-4 receptors on the plasma membrane ↑ from 30% to 65% between 6 and 16 days in vitro; from stimulation by NRG, PSD-95 also ↑. In addition, TACE in hippocampal cultures is resistant to cleavage, especially the JM-b isoform. Preclinical study

Coimmunoprecipitation Immunohistochemistry
Western Blot

Rats
The expression of PSD-95 ↑ in the medial PFC of maternal separation-exposed adolescents, while NR2A did not significantly ↑. This effect resulted in behavioral alterations.
The ↑ of NR2A in PFC is related to SCZ and acts largely through its association with the postsynaptic protein PSD-95.  The expression of Gpr85 was ↑ in multiple brain regions of the mice, while the mRNA levels of Gpr27 and Gpr173 ↓.
Gpr85 was colocalized with PSD-95 and Shank3 mice brain. The density of dendritic spines and their content of PSD-95, Syn, AMPA receptors, and BDNF ↓ in AI-injured rats. In addition, neural architecture was changed with thin, mushroom-shaped, stubby and broad spines.
In AI-injured animals, working memory and social behavior were altered, as well as PSD-95 and AMPA receptors.

Coimmunoprecipitation
Electrophysiology Western bot In HT mice, there was an ↑ in α1 subunit of the GABAA receptor and an ↑ in sIPSCs in mPFC pyramidal neurons, with a significant ↑ in evoked IPSCs, leading to excitatory-inhibitory dysregulation in such mice. PSD-95 deficiency enhances the functioning of inhibitory synapses through upregulation and trafficking of NLGN2 and reduction of GSK3β activity. In Shank3 TG mice several myelin-related genes were downregulated specifically in mPFC, but not in the striatum or hippocampus. mRNA levels of Gpr85 ↑ in multiple brain regions of the mice brain, while mRNA levels of Gpr27 and Gpr173 ↓ in cortex and striatum.

Shank and Psychotropic Drugs
Several studies have shown that antipsychotic treatments can affect the formation of the dendritic spine and synaptogenesis by direct rearrangement of the synaptic structure, regulating postsynaptic scaffolding proteins, such as Shank (Table 2). Atypical and typical antipsychotics are shown to differentially modulate the protein density at dendritic spines. Specifically, an in vitro study on rat hippocampal neurons demonstrated that clozapine administration increased Shank1a protein density along dendrites, whereas Shank1a protein density was decreased by haloperidol [102]. The differential modulation of Shank expression by clozapine and haloperidol may be consistent with a different impact on dendritic spine formation, suggesting the possible contribution of clozapine to higher efficacy in severe conditions, such as treatment-resistant schizophrenia [103]. Some authors have pointed out that chronic treatment with mood stabilizers, lithium, or valproate, used clinically as antipsychotic augmentation strategies in schizophrenia, can directly affect the expression of Homer1b/c and related Shank and inositol 1,4,5 trisphosphate receptors (IP3Rs) in cortical and subcortical regions [20,104]. In particular, Shank expression was affected by lithium in the cortex, whereas valproate was shown to downregulate gene expression in the motor and insular cortex [104]. However, lithium treatment significantly reduced IP3R in almost all cortical subregions, whereas valproate reduced it in the dorsolateral caudate-putamen [104]. Shank proteins interact with NMDARs through a PSD-95/GKAP complex and with mGluR1/5 by involving Homer1b/c, which via IP3Rs bind this machinery to intracellular Ca 2+ release [39]. Specifically, Homer1b/c, Shank, and the IP3Rs play an essential role regulating intracellular Ca 2+ oscillations with a potential neuroanatomical specificity, suggesting a differential intracellular Ca 2+ networks regulation by mood stabilizers [46,105]. Therefore, the impact of mood stabilizers' chronic administration on Shank and IP3R transcripts in cortical and subcortical regions might regulate calcium-dependent glutamatergic activity. Furthermore, the fact that Shank was modulated in brain regions specifically involved in cognitive, behavioral, and motor functions may further confirm its essential role in transductive pathways implicated in the mechanism of action of psychotropic drugs [104].          Post-mortem study qRT-PCR analysis Human brain tissue -There was a significant negative correlation with interneuronal markers, parvalbumin and somatostatin, while a positive correlation with spinophilin, but not with PSD-95.

PSD-95 Structure and Topography
PSD-95, also known as synapse-associated protein 90 (SAP-90), is the most abundant protein of the PSD [151,152], belonging to the membrane-associated guanylate kinase family (MAGUK). It is a scaffolding protein located at excitatory synapses and involved in the glutamatergic neurotransmission and trafficking of NMDARs and AMPARs [153,154]. PSD-95 presents three N-terminal PDZ domains (PSD-95, Dlg, and ZO-1), an src homology domain (SH3), and a guanylate cyclase domain (GUK) [152,155]. Through the PDZ domain, PSD-95 can directly connect with the C-terminus of NR2A and NR2B subunits of NMDAR and to AMPAR via stargazin/TARPs [156] while an indirect interaction is established with mGluR1/5 via GKAP [157][158][159][160]. Based on its involvement as a key component of PSD development and function, PSD-95 levels increase during development with maximum levels in adults, consistent with synaptic density growth in the human PFC [161]. Furthermore, PSD-95 localization is defined also through its post-translational modifications, such as palmitoylation [162,163] at Cys3 and Cys5 obtained by continuous palmitoylation/depalmitoylation cycles, necessary for its interaction with K+ channel KV1.4 and its clustering at the PSD [162,164,165].

PSD-95 Connectome and Signalosome
PSD-95 is an essential protein in the synaptic plasticity and dendritic spine morphogenesis during the neurodevelopment process, acting by the stabilization, recruitment, and trafficking of glutamatergic receptors [155,166,167]. Specifically, the PSD-95 effect on NMDAR localization and stabilization at the PSD is different depending on the period of life. In fact, it has been demonstrated in animal models that the expression levels of PSD-95 increase from early life to adulthood along with an elevation in NR2A-subunit levels and differently from SAP102, showing an opposite trend [161,168,169] (Table 1). Thus, during neurodevelopment until adult age, a critical switch from the SAP102/NR2B-NMDAR to PSD-95/NR2A-NMDAR complexes has highlighted the nodal role of PSD-95 as a mediator of NMDAR, consistent to prevent the hyperexcitability at the synapses [170,171]. The relevance of PSD-95 in the neurodevelopmental process is confirmed also by a recent study in which the reduced levels of the protein affect NMDAR and AMPAR function and levels in the medial prefrontal cortex (mPFC) during juvenile and adolescent periods. This study showed an increase in NR1 and NR2A, and a decrease in GluA1 subunit with behavioral alterations including lack of sociability, learning, and working memory deficits [171]. Concerning glutamatergic neurotransmission, PSD-95 is found to form a quinary complex including AMPAR, stargazin, serine racemase (SR), and the NMDAR [92]. SR binds to the third PDZ domain of PSD-95, while stargazin binds to the first and second PDZ domains [92]. AMPAR activation is responsible for the dissociation of SR from the complex and an increase in catalytic activity, favoring NMDAR transmission mediated by D-serine elevation [92]. The regulation of NMDAR by PSD-95 seems to be related also to a cytosolic sulfotransferase 4A1 involved in several neuropsychiatric disorders [172][173][174] that restore the correct processes of synaptic plasticity and the dendritic spine density [85,175,176]. PSD-95 also forms a complex with synaptosomal-associated protein 25kDa (SNAP-25) and p140Cap, opening the possibility that reduction in its levels may contribute to affect postsynaptic function and plasticity as demonstrated in SNAP-25 heterozygous mice with reduced densities of dendritic spines [87]. A decrease in PSD-95 levels is also reported in conditional heparin-binding EGF-like growth factor KO mice that are found altered in the brain and serum of patients affected by schizophrenia [177], underlining its crucial role in the pathophysiology of the disease [95]. PSD-95 is also involved in the regulation of inhibitory neurotransmission. In fact, transgenic adolescent mice models (PSD-95 +/− ) have shown a significant increase in the α1 subunit of GABA A in medial PFC with subsequent increases in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) in pyramidal neurons, responsible for a shift from excitatory to inhibitory balance [94]. In a preclinical study, Abbas et al. demonstrated that 5-hydroxy-tryptamine-(5HT) 2A -and 5-HT 2C -mediated downstream signaling were impaired in PSD-95 null mice, suggesting the role of PSD-95 in modulating and scaffolding macromolecular glutamatergic signaling complexes and metabotropic 5HT 2A and 5HT 2C receptors function [178]. PSD-95 is also directly connected to D1R and forms a ternary complex with Calcyon that regulates the entry of Ca 2+ by the membrane, playing a role in the internalization of D1R through its phosphorylation [179].

PSD-95 in Schizophrenia and Relevance for Antipsychotic Treatment
Several human and animal studies suggest that PSD-95 disruption is involved in the neuropathology of schizophrenia and ASD [180,181]. A recent preclinical study using mice with loss-of-function mutations in the PSD-95 protein complex and an innovative cognitive touchscreen test showed that distinct components differentially regulate learning, cognitive flexibility, and reaction times in cognitive processing. This study provided new insights into how mutations in this molecular complex could lead to different and complex phenotypes in neurodevelopmental disorders [182]. In addition, post-mortem studies have reported a reduction in PSD-95 mRNA and proteins in the dorsolateral prefrontal cortex (DLPFC) and dorsomedial prefrontal cortex of patients affected by schizophrenia compared with HC [126,183] (Table 2). A decrease in PSD-95 protein levels was found also in the cornu ammonis 1 of the hippocampus, a region that is critically involved in schizophrenia patients in comparison with matched controls [111]. These data are also confirmed by another post-mortem study where the PSD-95 expression was assessed in the frontal cortex, using immunoprecipitation and Western blotting in schizophrenia compared with HC, adding to existing evidence for disturbed postsynaptic glutamate function and synaptic plasticity in schizophrenia [134]. Furthermore, in preclinical studies conducted on mice, deletion in the gene Discs Large MAGUK Scaffold Protein 4 (DLG4) encoding for PSD-95, is associated with behavioral and molecular abnormalities, consisting of the clinical phenotype of repetitive behavior and irregular social interactions [184]. In addition, an immunoprecipitation study with Western Blot analyzed the scaffolding protein DLGAP1 localized in glutamatergic neurons, which is a component of protein complexes organized by PSD95. In this context, Dlgap1 KO mice exhibited disruption of protein interactions in PSD and sociability deficits, consistent with the findings of DLGAP1 haploinsufficient variants in schizophrenia and ASD [83]. Given the crucial role of PSD-95 in the structure and architecture of PSD, as well as in its signaling and function, several lines of evidence have demonstrated modulation by antipsychotics or psychotropic compounds that targeted these molecules [107,114]. Multiple preclinical studies have demonstrated their impact on PSD-95 mRNA expression. It has been demonstrated that an increase in dorsolateral and ventromedial caudate-putamen (p = 0.015 and p = 0.04, respectively) after memantine administration compared with MK-801, supporting the view that memantine differently modulates key PSD molecules contributing to the different activities on neuroplasticity exerted by this compound [114]. On the other hand, the administration of antipsychotics is involved in the modulation of PSD-95, in particular, asenapine, at doses of 0.05 mg/kg, 0.1 mg/kg, and 0.3 mg/kg, is responsible for increased mRNA levels in all cortical and subcortical regions assessed: anterior cingulate cortex (ACC), medial agranular cortex, motor cortex, somatosensitive cortex, insular cortex, dorsomedial caudate-putamen, dorsolateral caudate-putamen, ventrolateral caudate-putamen, ventromedial caudate-putamen, NAc, nucleus accumbens shell. Haloperidol at 0.5 mg/Kg is found to induce overexpression of PSD-95 in the ACC and motor cortex, while at the dose of 0.8 mg/Kg it is responsible for its increase in the ACC, motor cortex, somatosensory cortex, insular cortex, as well as in the caudate-putamen dorsolateral, dorsomedial, ventromedial, NAc, and nucleus accumbens shell [107]. Additionally, chronic administration of antipsychotics haloperidol and ziprasidone were evaluated at 90 min (early time-point) or 24 h (delayed time-point), where PSD-95 expression was found to be significantly increased in ventromedial caudate-putamen at the delayed time-point by ziprasidone compared with vehicle and by haloperidol in the striatum at the early time-point compared with delayed timepoint [119]. In this context, ziprasidone increased PSD-95 levels in the ACC at the early time-point whereas haloperidol is responsible for increasing the same mRNA in the medial agranular cortex, motor cortex, and somatosensory cortex [119]. D-aspartate-oxidase KO mice (Ddo −/− ), with persistently elevated brain D-aspartate levels, exhibited a decrease in PSD-95 expression in the motor cortex (p = 0.02), prelimbic cortex (p = 0.01), and in several following regions of caudate putamen-dorsomedial (p = 0.001), dorsolateral (p = 0.005), ventrolateral (p = 0.0003), ventromedial (p = 0.001) compared with wild type mice, suggesting that persistently elevated D-aspartate may cause complex adaptive mechanisms affecting striatal and cortical gene expression of key PSD transcripts by enhancing NMDA transmission [115]. Additionally, D-serine seems to be involved in modulating the interaction of postsynaptic SR with PSD-95, NMDAR, and other postsynaptic scaffold proteins as well as postsynaptic PSD-95 signaling. This interaction with nitric oxide synthase (nNOS) and synaptic AMPARs demonstrates the involvement of D-serine/SR with PSD-95 and NMDARs in glutamatergic synapse stability during synaptic development [137].

DISC1 Distribution, Structure and General Function
DISC1 is a susceptibility gene for schizophrenia and putatively for other psychiatric disorders, initially discovered in a Scottish family with a high frequency of mental disorders [179,[185][186][187][188]. DISC1 is a scaffold protein with a highly complex interactome at the PSD site and is involved in multiple brain functions relevant to the symptomatology and pathophysiology of schizophrenia [189]. Growing biological studies have indicated the relevance of DISC1 in early neurodevelopment and synaptic regulation, suggesting its role in determining multiple endophenotypes underlying major mental disorders [185]. Several reports have shown that common variants of DISC1 are associated with endophenotypes relevant to schizophrenia, including reduced gray matter volume and poor working memory [190][191][192][193]. Post-mortem immunoelectron microscopy studies have found that a pool of DISC1 was enriched in the PSD of the human brain [110], suggesting the relevance of DISC1 in synaptic activity. Even with the limitation of KO modeling, DISC1 KO mice may recapitulate some of the functional milestones associated with psychiatric disorders. In this framework, mice lacking DISC1 gene fast-spiking interneurons (INTs) showed reduced average firing rates of INTs with decreased spike transmission efficacy at local pyramidal cell (PYR)-INT connections in vivo, suggesting reduced excitatory responsiveness to local glutamatergic drive as a potential mechanism of lower INT rates [194]. The DISC1 KO mice might be considered a proxy of prodromal schizophrenia pathophysiology relevant for studying psychotic conversion from the prodromal to the full-blown psychosis phase, suitable for early intervention [195]. In this context, the chronic administration of N-acetylcysteine (NAC) has been shown to reduce the amphetamine-induced hyperlocomotion and revert the subcortical "dopamine storm" in animal modeling by acting on dopamine receptors, expression of glycogen synthase kinase 3 (GSK3), medium spiny neuron (MSN) dendritic impairments, and striatal parvalbumin (PV) density, demonstrating the preventive effect of chronic NAC treatment. These findings supported the benefit of NAC as a dietary supplement for schizophrenia prodromes but also the relevance of striatal dopamine receptors, PV neurons, and GSK3 signaling pathways as therapeutic targets for treating or preventing the pathogenesis of mental disorders [195].
Beyond PSD localization, DISC1 has been detected in the cytoskeleton along with actin filaments and girdin, which, via kinesin-based transport along the cytoskeleton, might play a role in axonal elongation [196,197]. Of interest, it has been hypothesized that truncated DISC1 might be responsible for microtubule disruption and a disorganized microtubule network [196,198,199]. DISC1 has also been localized to growth cones, detected in the hippocampus, interacting with multiple binding partners [197,199,200], such as the centrosome, which is involved in the recruitment of kendrin, dynein, and dynactin subunits, lissencephaly 1, nuclear distribution protein E homolog-like-1 (NDEL1), pericentriolar material 1 (PCM1), ninein, and coiled-coil protein associated with myosin II and DISC1. The centrosome has also been shown to interplay with cAMP-specific 3 ,5 -cyclic phosphodiesterase 4B/4D (PDE4B/PDE4D), nuclear distribution protein E homolog 1, or nuclear distribution element 1 (NDE1), and Bardet-Biedl Syndrome 4, and these interactions have been implicated in microtubular asters formation, neurite outgrowth, and neuronal migration [198,[201][202][203][204][205][206][207]. DISC1 has also been shown to localize to several subcellular compartments, including cilia, which have been implicated in the regulation of their formation and maintenance. In this regard, it has been reported that the knockdown of DISC1 induced a marked reduction in primary cilia and established subtype-specific targeting of dopamine receptors on the ciliary surface, suggesting a relationship between DISC1 and neural dopamine signaling [208]. Further localization of DISC1 has been found on membrane fractions, where it has been shown to interact with amyloid precursor protein (APP), crucial in the formation of cortical precursors [209][210][211], and with mitochondria present on microtubules, regulating their "ring" structures and trafficking. Moreover, interaction with mitofilin within mitochondria has been reported to be critical for the proper functioning of the electron transport chain, monoamine oxidase, and Ca 2+ activity [212][213][214][215].

DISC1 Connectome and Signalosome
DISC1 is a molecular partner in a large interactome comprising more than a hundred binding molecules, which acts as a structural and functional protein regulating multiple and parallel signaling pathways implicated in neurodevelopment diseases [216]. The function of DISC1 has been studied in human induced pluripotent stem cells (iPSCs) inoculated with an endogenous 3X-FLAG tag at the C-terminus of canonical DISC1 using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) [187,[216][217][218][219]. In this context, DISC1 has been proven to act in a cell type-and contextdependent fashion, relevant to the neural development and integration of psychiatric disease risk factors within a set of defined molecular functions [216]. Indeed, DISC1 has been shown to regulate cell division, metabolism, and synaptic function based on its location and protein interaction in cytoplasmic compartments, such as the kinetochore, mitochondria, and pre-and postsynaptic sites, respectively [44,185,220,221]. However, most of the connections identified in the interactome have not yet been fully validated, and the functional outcome of these interactions remains to be elucidated.
Preclinical animal model studies in which DISC1 isoforms were genetically deleted demonstrated morphological changes in the dendritic spine [222]. The role and specificity of DISC1 in modeling the dendritic spine were elucidated after the discovery of its binding to Kalirin7 (Kal7), a key regulator of the dendritic structure, preventing the interaction of Kal7 with Rac1, GTPase protein that is highly reactive to NMDAR activation by glutamate, thereby reducing NMDAR/NR2-dependent signaling [223] (Figure 2) (Table 1). This mechanism, mimicking disturbances in glutamatergic neurotransmission, has been frequently reported in schizophrenia, leading to the disruption of dendritic spines, and resulting in major pathological changes in brain function. Furthermore, the concept of a signalosome involving disease-associated factors, such as DISC1 and glutamate, may contribute to the multifactorial and polygenic features of schizophrenia [223]. Several lines of evidence have suggested a putative role for DISC1 in regulating fundamental events in neural development, from precursor cell proliferation and differentiation to synaptic specialization and function. Although the mechanisms leading to the regulation of gene expression are unknown, studies in mouse hippocampal neurons [224] have shown that DISC1 regulates the transcriptional activity of activating transcription factor 4 (ATF4), also known as cAMP-responsive element binding protein-2 (Creb2) [225], contributing to tumor formation. Of interest, ATF4 has been identified as a transcription factor linked to synaptic processes associated with cognitive function, and DISC1 is its most abundant binding partner in the nucleus of mutant human neurons, showing an increased connection to multiple dysregulated genes [224]. In this regard, preclinical studies identified a binding region between DISC1 and ATF4 in the genomic locus of PDE4D, a gene implicated in psychiatric disorders [226]. The results of this study showed that loss of DISC1 or ATF4 function increased the transcription of PDE4D9. Considering that PDE4D9 is increased by dopaminergic stimulation of D1-type dopamine receptors [227,228], the study results suggested that DISC1 release mediated by transcriptional repression of PDE4D9 might act as a regulator of feedback inhibition of dopaminergic signaling [224]. Considering the involvement of DISC1 in many neural functions, such as neurogenesis, neural differentiation, migration, and neurotransmitter signaling, how can changes in DISC1 structure and function affect the dopaminergic transmission, and how can they interfere with the pathophysiology of schizophrenia? Gathering together the long-recognized alterations in dopaminergic neurotransmission in schizophrenia, a surprising impact of DISC1 on dopaminergic function has been elucidated with increasing attention to its role in the dopamine D2 receptor (D2R) regulation and the response to antipsychotics [229]. In this regard, DISC1 has been shown to form a protein complex with D2R, the principal pharmacological target of antipsychotics. The D2R-DISC1 complex has been found in the brain tissue of patients with schizophrenia, promoting GSK3 signaling and inhibiting D2R internalization (Figures 2 and 3). Su and colleagues demonstrated that the DISC1 variant R264Q, located within the D2R-binding region, was shown to increase DISC1 affinity for D2R by promoting GSK3 activity, suggesting a possible mechanism by which this common polymorphism could influence aspects of brain function relevant to schizophrenia, which might be explored for the development of novel drug treatments [229]. Concerning other neurotransmitter signaling pathways in which DISC1 is implicated, it should be noted that DISC1 heterozygous mutant mice show elevated levels of PDE4 associated with synaptic abnormalities in the cortex, hippocampus, and the whole brain, with social and cognitive behavioral deficits [230]. In this framework, DISC1 has been shown to bind PDE4 family members that negatively regulate adenosine 3 ,5 -cyclic monophosphate (cAMP) signaling, negatively influencing synaptic plasticity [231] (Figure 2). In this view, the PDE4 pathway may represent pathological molecular signatures in human neurons derived from patients with schizophrenia and other major mental disorders and potentially considered a target for drug development. An intriguing relationship between PDE4 signaling and the DISC1 mutation has been documented with potential therapeutic implications. Specifically, cortical neurons differentiated from iPSCs derived from patients with schizophrenia and carrying a deletion in the DISC1 gene showed a reduction of synaptic vesicle protein (SV2 + ) puncta number after four weeks of neural differentiation [232], associated with an increase in PDE4-related signaling [230]. Some studies showed that pharmacological inhibition of PDE4 has proven to restore synaptic transmission and social and cognitive behavioral alterations [233,234]. Collectively these findings suggested that dysregulation of PDE4-cAMP signaling might be a potential convergent patho-molecular mechanism underpinning schizophrenia and other mental disorders and could provide a target for future drug development [230].

DISC1 and Schizophrenia: Human Common and Ultrarare DISC1 Variants
A growing number of biological studies have focused on the DISC1 gene, a wellestablished genetic risk factor in a spectrum of psychiatric disorders, supporting the connection between common and ultrarare DISC1 variants and their structural and functional implications associated with major mental illnesses [187]. Three common DISC1 gene variants associated with behavioral disorders have been discovered: L607F, S704C, R264Q [193,235], and several rare and ultrarare variants: G14A, R37W, S90L, R418H, T603I, S209R, R338Q, T754,S and P758R [236][237][238]. It has been reported that the common variants S704C and R264Q may slightly increase the risk of developing schizophrenia, whereas the rare and ultrarare variants are associated with a 2% risk of schizophrenia [193]. Reduced gray matter in the superior frontal gyrus, anterior cingulate cortex, and left supramarginal gyrus was found in F607 allele carriers compared with L607 homozygotes, as well as suffering from more severe positive symptoms in schizophrenia, providing a potential mechanism by which DISC1 could confer an increased risk for schizophrenia [239,240]. Of interest, a recent report showed that schizophrenia patients

DISC1 and Schizophrenia: Human Common and Ultrarare DISC1 Variants
A growing number of biological studies have focused on the DISC1 gene, a wellestablished genetic risk factor in a spectrum of psychiatric disorders, supporting the connection between common and ultrarare DISC1 variants and their structural and functional implications associated with major mental illnesses [187]. Three common DISC1 gene variants associated with behavioral disorders have been discovered: L607F, S704C, R264Q [193,235], and several rare and ultrarare variants: G14A, R37W, S90L, R418H, T603I, S209R, R338Q, T754,S and P758R [236][237][238]. It has been reported that the common variants S704C and R264Q may slightly increase the risk of developing schizophrenia, whereas the rare and ultrarare variants are associated with a 2% risk of schizophrenia [193]. Reduced gray matter in the superior frontal gyrus, anterior cingulate cortex, and left supramarginal gyrus was found in F607 allele carriers compared with L607 homozygotes, as well as suffering from more severe positive symptoms in schizophrenia, providing a potential mechanism by which DISC1 could confer an increased risk for schizophrenia [239,240]. Of interest, a recent report showed that schizophrenia patients with severe positive symptoms who do not respond to antipsychotic treatment showed a reduced superior frontal metabolism [241].
In particular, in vitro studies have found that the F607 variant is associated with a reduced release of noradrenaline, and a reduced ability of DISC1 to interact with the PCM1 protein of the centrosome, resulting in defects in mitochondrial trafficking [214,242]. Furthermore, carriers of the F607 allele also express lower levels of a DISC1 splice variant expressed in patients with schizophrenia [243]. The S704/C704 variant has been associated with structural (including volume and integrity of gray matter) [244,245] and functional abnormalities of the hippocampal and para-hippocampal regions [190,246], severe positive symptoms in patients with schizophrenia [247], and further DLPFC activity during a working memory task [248]. Studies on the effects of R264Q have been limited to a modest impact on cortical thickness in the lateral occipital gyrus [239] while evidence of modifications caused by L607F and S704C has emerged [236]. At the molecular level, as with L607/F607, the 704 amino acid variation has also been implicated in altered PCM1 localization and DISC1 expression [243,249]. In addition, the C704 allele has been associated with reduced activity of extracellular signal-regulated kinase 1 (ERK1) and serine/threonine kinase RAC-alpha (AKT) (Figure 2), altered DISC1 binding affinity for NDE1 and NDEL1, and a change in DISC1 oligomeric status [206,244,250,251], relevant for schizophrenia.

DISC1, a Drugable Molecule?
DISC1 is a multifunctional scaffold protein that interacts with several proteins involved in neuronal migration, neurite outgrowth, cytoskeleton modulation, and signal transduction [252] whose interactome has been an attractive target for potential therapeutic interventions [253]. Chronic administration of the atypical antipsychotics olanzapine and risperidone has been shown to increase DISC1 mRNA expression in the frontal cortex of mice, whereas haloperidol and clozapine had no effect (Table 2). Furthermore, olanzapine also has been shown to increase DISC1 mRNA expression in the hippocampus similar to risperidone [254] (Figure 3). In clinical trials, DISC1 missense variants have been found in patients affected by schizophrenia with a form of ultra-resistance to treatment, suggesting that DISC1 variants may have a functional influence on the mechanisms of action of antipsychotics and treatment response [255]. In this regard, it has been reported that repeated oral administrations of clozapine, but not haloperidol, in the dominantnegative form of disrupted-in-schizophrenia 1 (DN-DISC1) transgenic mice treated with polyinosinic:polycytidylic acid (polyI:C), were able to improve cognitive impairment in the animal model. This study postulated that the subsequent increase in DISC1 may impact modulation of synaptic spines as a potential mechanism of action of the atypical antipsychotics [256] (Figure 3). The main reasons currently hindering the development of an effective small molecule targeting DISC1 are mainly the lack of knowledge of the multiple forms of splicing, 3D structure, and a large number of interacting proteins (about 200) [12,187]. The potential impact on AKT signaling and the growing evidence for a role in dopaminergic and glutamatergic signaling have placed the DISC1 pathway as a promising but challenging target for future pharmacological development [187].

Homer1: Distribution, Structure, and General Function
A multifunctioning and highly interacting class of proteins at the PSD, the Homer1, were discovered and cloned in a series of experiments reputed to unveil the molecular determinants of mGluR type I signaling [257] at a glutamatergic synapse [12,258,259]. Even if detected in several different tissues (i.e., skeletal muscle, heart), the largest distribution is the cerebral one. In the brain, the highest concentration has been reported (in decreasing order) in the striatum, cortex, and the hippocampus [260][261][262][263]. At least two isoforms of Homer1 proteins have been described: constitutive (Homer1b/c, Homer2a/b, Homer3) and inducible (Homer1a and Ania-3). The long form Homer1b/c is characterized by enabled/vasodilator-stimulated phosphoprotein homology (EVH1) sequence at the N-terminus, and a coiled-coil forms at the C-terminus that allows the single molecules of Homers to interact with each other. It forms a sea-star-like macromolecule, whose free-standing EVH1 terminal may interact with multiple intracellular targets included among others: PSD proteins (i.e., PSD-95, Shank1) [8,260,[264][265][266]. Homer1 plays an important role in working memory [267,268], executive functions [269], and regulation of cortical hyperexcitability, and it has been hypothesized to play a pivotal role in dopamine-glutamatergic neurotransmission, relevant in the pathogenesis of schizophrenia [270][271][272]. Moreover, Homer1 has multiple functions including modulation of mGluR type I trafficking, regulation of Ca 2+ signaling, control of LTP, growth of dendritic spines, and control over synaptic plasticity [8].

The Homer Connectome and Signalosome
Homer1s are capable of dispatching intracellularly multiple convergent or divergent signaling, this feature may be the result of the different isoforms, the constitutive or immediate early-gene related functions, as well as the sophisticated Homer interactome [8,273]. Homer1-interaction partners are reported in UniProt [274]. In fact, among the PSD proteins, Homer1 is a key component of the dendritic spine and a regulator of neuronal signaling [275][276][277]. A principal implication of Homer1 in cellular signaling is the modulation of glutamatergic transmission mainly related to mGluR type I, due to the role of glutamate in schizophrenia pathophysiology makes the Homer1 family of potential relevance both for the structure and function of the dendritic spine at a glutamatergic synapse ( Table 1). The assembling/disassembling of Homer1 and its interacting partners is probably the most evident consequence of glutamate transmission effects on Homer-related signal transmission. One interaction that has been recently highlighted for a robust association between glutamate function and schizophrenia is the one between Homer1 and insulin receptor substrate type53 (IRSp53), aka BAR/IMD domain containing adaptor protein 2 (BAIAP2). The latter is a scaffold protein located at the PSD of glutamate synapse that bridges membranes with the cytoskeleton in actin-filled protrusions and is believed to be implicated in synapse development. In addition, the IRSp53 and Homer1 interaction is probably mediated through PSD-95 [278]. The induction of Homer1a by synaptic stimuli impacts intracellular Ca 2+ oscillations through the regulation of the coupling of long Homers with IP3R, transient receptor potential channels (TRPCs) (Figure 2), and ryanodine receptor (RyR) [279].

Homer, Schizophrenia, and Antipsychotic Treatment
Three studies have investigated Homer's expression in the post-mortem brain of schizophrenia patients with a focus on the cortex and/or hippocampus. A decrease in Homer levels was detected in the cortex and hippocampus of schizophrenia patients compared to HC with no separation between the two major isoforms [11]. A study conducted on the CA1 region of the hippocampus (20 schizophrenia patients and 20 HC brains) has shown a reduction in the protein levels of the long form Homer1b/c (−43%) and an increase of the short form Homer1a (+25%) with a ratio Homer1a/Homer1b-c >1 [111]. Changes were also reported in several proteins interacting directly or indirectly with Homer1-included PSD-95 [280]. Finally, utilizing products determined with denaturing high-performance liquid chromatography and analyzed by polymerase chain reaction, it was demonstrated that the allele frequency of single nucleotide polymorphisms (SNPs) in a DNA pool of 368 schizophrenia patients and 368 HC, Homer1-IVS4+18A>G (statistically significant with individual genotyping, p = 0.01) were associated with schizophrenia, identifying a plausible candidate for the phenotype of the disease [281]. Preclinical studies in rats have shown that Homer expression was changed by the administration of different antipsychotics (Table 2). Specifically, Homer1a was significantly induced by haloperidol in dorsomedial, dorsolateral, ventromedial, ventrolateral caudate-putamen, and NAc [106,108,112], and by asenapine and olanzapine in dorsolateral caudate-putamen [112]. Furthermore, olanzapine induced Homer1a gene expression in the caudate-putamen regions and in the nucleus accumbens shell, whereas asenapine induced it in the ACC, medial agranular cortex, motor cortex, dorsolateral caudate-putamen, and NAc [112,113]. Homer1a was induced by tergulide in ventrolateral caudate-putamen [118]. Additionally, tergulide also reduced Homer1b in the ventrolateral caudate-putamen, ventromedial caudate-putamen, and shell of NAc [118]. Preclinical studies demonstrated that ketamine increased Homer1a expression in the insular cortex, and decrease Homer1b in the motor cortex, dorsolateral caudate-putamen, ventromedial caudate-putamen, and medial agranular cortex [114,117]. In acute administration of clozapine, Homer1a was increased in all caudate-putamen subregions, in the NAc, and medial agranular cortex. In addition, clozapine and ziprasidone both induced Homer1a in somatosensory cortex, and insular cortex. The chronic administration of ziprasidone increased Homer1a only in the NAc and dorsolateral and ventrolateral caudate-putamen, and even induced Homer1b in dorsolateral caudate-putamen. During chronic clozapine treatment Homer1a expression was induced only in the NAc, suggesting that Homer1a preserves its expression profile after chronic antipsychotics [119]. Haloperidol also induced Homer1b in the ACC, motor cortex, medial agranular cortex, and insular cortex. Furthermore, Homer1b/c expression was increased more by asenapine than by haloperidol in the insular cortex and somatosensory cortex [116]. In this context, the impact of amisulpride on gene transcripts when administered in naïve rats compared with rats pretreated with antipsychotics demonstrated differential effects in the cortex and striatum by increasing Homer1a levels in the NAc nucleus and inducing Homer1b/c in striatal regions more than haloperidol [116]. These results are suggestive of a differential neurobiological impact on PSD transcripts induced by antipsychotics.

The Role of PSD Transmembrane AMPAR Regulatory Proteins in Schizophrenia: Stargazin
The transmembrane AMPAR regulatory proteins (TARPs), such as stargazin, were demonstrated to be crucial for the AMPAR subunits, regulating their both receptor trafficking and gating [153] (Figure 2). Stargazin showed a positive allosteric modulation of AMPARs, promoting the AMPAR surface expression and localization, exerting a regulatory role of intracellular channel conductance [282]. On the surface of the PSD, AMPAR binds to stargazin, connecting the intracellular proteins containing PDZ domains, such as PSD-95, stabilizing the activated state of AMPAR [283,284]. Indeed, in neurons that do not express stargazin, the majority of AMPARs fail to reach the dendritic surface and a substantial proportion of AMPARs, which are intracellularly retained, exhibit immature glycosylation crucial for the efficacy of synaptic transmission in the brain [285]. Preclinical studies on hippocampal slices suggested that synaptic recruitment and activation of AMPAR are regulated by PSD-95 via stargazin, which is involved in the extra-synaptic expression of AMPAR [286] and interactions with PSD-95 for stabilization and NMDARdependent trafficking [287]. Several authors have pointed out that LTP and long-term depression (LTD), the most extensively studied forms of long-lasting synaptic plasticity regarded cellular mechanisms underlying learning and memory, are associated with AM-PAR recruitment [288] and trafficking [289]. Preclinical studies have reported that while showing normal localization of NMDARs, PSD-95 mutant mice exhibit deficits in LTP, LTD, and spatial learning, suggesting that PSD-95 may play a critical role in coupling NMDARs to downstream transduction pathways [290]. Furthermore, PSD-95 also appears to be indirectly linked to AMPARs through stargazin interaction [286]. The bidirectional interconnection between PSD-95 and stargazin mediates the targeting of AMPARs to the synaptic membrane, and, therefore, it is conceivable that a functional link between NM-DARs and AMPARs occurs via the PSD-95-stargazin complex, potentially dysregulated in the pathophysiology of schizophrenia. In a recent post-mortem study of patients with schizophrenia, Benesh and collaborators suggested that associations between stargazin and AMPA subunits are abnormal, potentially affecting the forward trafficking or synaptic stability of subunit of glutamate GluA1-containing AMPARs. This study provides evidence of altered interactions with trafficking proteins that contribute to glutamate dysregulation in schizophrenia [291]. AMPARs and their modulators, such as TARPs, mediate most of the fast synaptic transmission in the mammalian brain and spinal cord, representing attractive targets for therapeutic strategies [92], also with a role as anticonvulsants or neuroprotectants [292] (Table 1). Moreover, the trafficking and gating properties of AMPARs may be amplified by these TARP proteins expressed in localized brain regions, including the forebrain and hippocampus [293]. However, bioavailability could be a frequent failure of small molecules that target PSD proteins, such as TARPs, that amplify AMPAR pharmacology by altering the rapid excitatory properties of glutamate [12]. Of interest, the potential strategy of adding compounds to antipsychotics that modulate TARPs requires further investigation as a possible approach in schizophrenia treatment [12]. Remarkably, genes encoding for PSD proteins have been considered promising candidates for the identification of new treatments in diseases where PSD dysfunction underpins a genetic framework of vulnerability, such as schizophrenia. It should be noted that the calcium channel gamma-2 subunit gene, which codes for stargazin, has been considered a pharmacological target gene [294]. In light of this, several findings have contributed to highlighting the association of PSD genes related to neurodevelopmental disorders (especially schizophrenia and ASD), including whole-genome analyses linking multiple TARP loci to childhood epilepsy, schizophrenia, and bipolar disorder where, chaperones AMPA receptors emerge as crucial in excitatory synapses, implicating both in signal transduction and in neuropsychiatric disorders [295]. As a matter of fact, recent GWAS findings, proteomic, post-mortem, neuroimaging, and genetic analyses, as well as preclinical models, have suggested a relevant role of PSD genes in the pathophysiology of schizophrenia [8,11,23,24,171,296,297].

Arc Distribution, Connectome, and Signalosome
The activity-regulated cytoskeleton-associated protein (Arc) (also known as Arg3.1, and KIAA0278) is an immediate early gene (IEG) encoding for a protein consisting of a coiled-coil domain at the N-terminus, multiple endocytic protein binding domains, and a homology domain with spectrin, a cytoskeletal protein, at the C-terminus [298]. Arc gene presents a DNA sequence normally found in the RNA of the human immunodeficiency virus, responsible for forming virus-like particles [299]. Thanks to its structure, Arc mRNA is able to link Arc protein and form a capsid containing Arc mRNA. These structures are encapsulated in the extracellular vesicles that cross the neuron membrane to other cells, such as microglia or other neurons, in a spatial/temporal specific manner, relevant for Arc activity, including synaptic plasticity, learning, memory consolidation, and behavior experiences [300][301][302]. At the PSD site, Arc mRNA localizes in the PSD-95-NMDAR complexes with kinesin motor complex and other proteins including fragile X mental retardation protein (FMRP) and Pur-alpha [303]. At this site, Arc interacts directly with an inactive form of CaMKIIβ targeting the spine as actin-rich dendritic spines, relevant for synaptic plasticity [304] (Figure 2). Given its centrality in synaptic architecture, Arc is highly conserved in vertebrates and is specifically expressed in the nucleus and dendrites, and at the PSD of cortical and hippocampal glutamatergic neurons [305]. Arc is the only IEG that moves through the dendritic arborization after its expression, differently from the others that remain localized in the cell body [306]. Its transcription is inhibited by AMPAR, which regulates its levels after about five minutes of its induction by various stimuli, including seizures in the hippocampus, LTP, and LTD [307][308][309]. The C-terminus of Arc is involved in the regulation of cytoskeleton structure via interaction with F-actin [305], microtubules, and microtubule-associated protein 2 [310]. Furthermore, Arc is able to maintain the phosphorylation of cofilin, the actin depolymerization factor, resulting in an increase of F-actin [311]. This process is responsible for an increase in the proportion of thin and filopodia-like protrusions and density of spines in neurons of the hippocampus as demonstrated in a comparison of wild type and Arc KO mice [312]. Concomitantly, mature and mushroom-shaped spines are found in the same animal model indicating a plausible negative influence of Arc on spine maturation [312]. Moreover, a decrease in spine density seems to be related to Arc aberrant expression related to NMDAR hypofunction [70], emphasizing its role in regulating dendritic spine density and morphology [313], relevant for schizophrenia ( Table 1). The link between Arc expression and glutamate neurotransmission seems to be related also to AMPAR function through its association with Arc's endocytic protein-binding domains [298,314]. In fact, Arc is also involved in modulating AMPAR trafficking through its translocation into the nucleus by regulating transcription and homeostatic plasticity [314][315][316][317]. On the other hand, NMDAR, mGluR type I, Tropomyosin receptor kinase B, and muscarinic acetylcholine receptor promote Arc transcription, converging on protein kinase A (PKA), protein kinase C (PKC) [318], and ERK [319].

Arc in Schizophrenia and Antipsychotic Modulation
Changes in Arc expression seem to be associated with several mental disorders, including major depressive disorder and schizophrenia [320][321][322]. Furthermore, aberrant expression of Arc could be relevant for cognitive impairment, fragile X mental retardation syndrome, as well as its reduced ubiquitination may occur in Angelman syndrome [322][323][324]. Kirov and colleagues, using a systematic analysis of synaptic protein complexes merging with novel copy number variants (CNVs) datasets, identified de novo mutations in the PSD proteome by enrichment for NMDAR and the Arc protein complex [89]. This study indicated that defects in postsynaptic signaling of NMDAR and Arc might play a significant role in the pathogenesis of schizophrenia [89]. Additionally, Arc is involved in the interaction between γ-secretase and trafficking endosomes relevant for the processing of APP to β-amyloid peptide, resulting in an increase in the levels of the latter related to Alzheimer's disease [325][326][327][328]. Concerning the neurobiological basis of schizophrenia, several preclinical studies have demonstrated the involvement of Arc in dopamine-glutamate interaction within the PSD and antipsychotic modulation, relevant for synaptic plasticity [329][330][331] (Figure 3).
Both typical and atypical antipsychotics can modulate Arc expression [332,333]. In this regard, typical antipsychotic haloperidol is responsible for an Arc increase in the NAc and striatum after 1h from the treatment, while the atypical antipsychotic clozapine reduces Arc levels in the thalamus and hypothalamus after 6h of treatment [334]. Another study evaluated the effects of the same drugs after acute and chronic treatment on the Arc protein levels showing an increase in the caudate after haloperidol administration in both settings but no effect after clozapine administration [335]. Furthermore, haloperidol increased its levels in the NAc core following acute treatment different from clozapine, which increases its levels in the shell of the NAc after acute treatment [335]. Clozapine was shown to decrease Arc protein levels in the mPFC and cingulate cortex after both acute and chronic administration [335]. Haloperidol induced also an increase of Arc in the whole striatum [106,107] except for the NAc shell compared with vehicle and sertindole [109]. A preclinical study demonstrated Arc increased levels in the striatum at 30, 60, and 120 min after haloperidol injection, while olanzapine was responsible for an increase only at the 30 min time point. At the same time, both haloperidol and olanzapine reduced Arc levels in the frontal cortex at 60 min [336]. Further preclinical evidence showed that haloperidol significantly increased Arc levels compared with amisulpride in the striatum [333]. Additionally, caffeine can modulate the effect of Arc mRNA and protein levels induced by haloperidol; in fact, the coadministration of caffeine and haloperidol reduced the Arc expression in the striatum and cortex compared with haloperidol [108] (Table 2). Finally, pretreatment with atypical antipsychotics 1 h before the administration of phencyclidine (PCP), which mimics psychotic symptoms in murine animal models, significantly reduced the Arc PCP-induced increase in the mPFC and NAc, suggesting a crucial role of antipsychotics in Arc modulation, relevant for therapeutic effects [337].

Neuregulin
The trophic factor Neuregulin 1 (NRG1, also known as heuregulin1 or neu differentiation factor) is a member of the epidermal growth factor (EGF) family which is involved in the inhibitory control of executive functions including working memory [338,339]. NRG1 is one of the four members of the NRG gene family characterized by an EGF-domain whose target is represented by ErbB receptors implicated in learning, memory, and other higher brain functions [340]. Multiple lines of evidence have pointed to the NRG1-ErbB4 signaling in parvalbumin-positive (PV+) basket cells as responsible for the reduction of cortical excitability [341] through the stimulation and release of the inhibitory transmitter γ-Aminobutyric acid (GABA) [342]. The NRG1 EGF-like domain is involved in the regulation of gamma oscillations [338], pyramidal cell excitability [339], seizure activity [343][344][345], and synaptic plasticity [346][347][348][349]. In addition, NRG is also responsible for the regulation of neural synchrony of local field potentials in the ventral hippocampus and PFC [350]. The activation of ErbB4, through the NRG1-EGF-like domains mediated phosphorylation, results in the activation of several intracellular cascades relevant for the incorporation of AMPA receptors into postsynaptic membranes and NMDAR regulation in interneurons, potentiating excitatory neurotransmission as demonstrated by preclinical study [57,80,123,[351][352][353]. Therefore, the enhanced stimulation of ErbB4 on the cell surface could be responsible for increased downstream molecular signaling at the PSD level, facilitating receptor dimerization and efficient NRG signal transduction [78]. NRG signaling is also directly related to PSD-95, increasing its levels near excitatory synapses, and regulating synaptic molecular composition through stimulation of ErbB4 receptors and the subsequent activation of mitogen-activated protein kinase (MAPK) [80,122,352,[354][355][356][357][358]. Specifically, the NRG1/ErbB4 pathway was seen to interact with PSD-95 and NMDAR, and that chronic administration of MK-801, induced a short-term potentiation of the functional interaction between NRG1/ErbB4 and NMDAR in the PFC and hippocampus of the rat, along with anxiety-like behavioral abnormalities, providing further evidence for cross-talk between the two signaling pathways [79]. Furthermore, Shi and collaborators recently proposed that the downregulation of the NRG1-ErbB4-mediated PV+ interneuron signaling could reactivate cortical plasticity for information processing, attention, and cognitive flexibility severely impaired in neurodevelopmental psychiatric disorders, including schizophrenia [340]. Furthermore, another mechanism of NRG1/ErbB4 interactions to promote dendrite growth in mature interneurons is mediated by Kal7, whose encoding gene KALRN has been associated with schizophrenia [75] [359] (Table 1). Moreover, a recent study evaluated several proteins of PSD and discovered 10 rare missense mutations in PSD protein-related genes, including NRG1 as susceptibility factors for schizophrenia [10] (Table 2). These data are coherent with several preclinical studies pointing to the involvement of the NRG1/ErbB4 pathway in the pathophysiology of schizophrenia as demonstrated by the perinatal administration of phencyclidine (PCP) to rats, which models schizophrenia-like symptoms [124]. In particular, the NMDA receptor antagonism exerted by PCP in the early phases of neurodevelopment may affect NRG1/ErbB4 expression [124]. Of interest, it has been shown that ErbB4 may modulate extracellular dopamine through the p38 MAPK signaling pathway, suggestive of the role of NRG in regulating the dopamine pathway relevant as a pharmacological target for psychotic disorders [358].
8.4. Synapse-Associated Proteins Family 8.4.1. Synapse-Associated Proteins Spatial and Temporal Distribution at the PSD Dlg or SAPs are scaffold proteins, members of the MAGUK superfamily, which exert essential functions in the regulation of synaptic plasticity and metaplasticity processes [360]. As all the MAGUK proteins, SAPs are characterized by the co-expression of an Src homology 3 (SH3) domain, one to three PDZ motifs, and a GUK domain which lacks catalytic activity and is mainly involved in mediating protein-protein interaction [360]. Structural differences have been highlighted between SAPs with SAP102 lacking the N-terminus cysteines and L27 domains that are required for the synaptic clustering of SAP-90/PSD-95 and SAP97 [361]. Otherwise, the SH3 and GUK, but not PDZ, domains are required for SAP102 stabilization at spines [74]. SAP102 and SAP97 are encoded by DLG3 and DLG1 genes, respectively, and both molecules are expressed at the PSD level, where they serve as a bridge between membrane receptors and the intracellular cytoskeleton [362,363].
Even though it is well-defined that MAGUKs represent key components at the neuronneuron interface, the specific SAPs interactome is highly challenging to define due to the presence of multiple motifs and, thus, several interaction partners, including scaffold proteins, cytoskeleton components, voltage-dependent channels, and neurotransmitter receptors [364][365][366][367]. However, significant interactions have been detected between the PDZ domains of SAP102 and SAP97 with the C-terminus tail of the NMDAR subunits NR2B and NR2A, respectively [362,368]. The different interactome exhibited by SAP102 and SAP97, together with a different time-dependent expression pattern, accounts for different functional tasks in terms of PSD organization and downstream signaling cascades, as discussed below. MAGUKs have been implicated in the time-dependent clustering of glutamate and other neurotransmitter receptors during the neurodevelopmental period as well as in the setting of LTP and LTD [364]. SAPs have been shown to regulate NMDAR trafficking and membrane expression by the direct interaction between NR1 and NR2 subunits with MAGUKs' PDZ domains [364]. Moreover, the membrane surface exposure of NMDAR subunits has been found to depend on SAPs temporal patterns of expression [369]. Specifically, SAP102 expression is more pronounced during the early neurodevelopmental phases and decreases rapidly in later stages, concurring with an increase in SAP90/PSD-95, PSD-93, and SAP97 levels [369]. Along with these changes, the shift from SAP102 to PSD-95 at the PSD couples with a reduction in NR2B expression and an increase in NR2A levels [369]. In fact, NR2B has been demonstrated to preferentially interact with SAP102 whereas NR2A better binds to PSD-95 [369]. The switch between different patterns of expression of scaffold proteins and NMDAR subunits is required for fine-tuning the plastic processes that occur during the neurodevelopmental period probably by differently recruiting signaling molecules necessary for proper synaptogenesis and spines maturation [370]. SAPs proteins have also exhibited the capability to differently regulate intracellular signaling pathways. Of interest, Ca 2+ /CaMKII has been shown to bind and phosphorylate SAP97, modulating AMPAR activity [371]. Otherwise, SAP102 is essential to couple NMDAR to the MAPK/ERK pathway [372] (Figure 2). In fact, SAP102 KO mice displayed an elevation of phospho-ERK protein levels, which were attenuated in response to NMDA stimulation, and responsible for impairment in LTP processes, resulting in cognitive alterations of flexibility and spatial learning [372].

Synapse-Associated Proteins in Psychotic Disorders: Clinical and Pharmacological Implications
Considering their relevant role in the organization of the PSD and NMDAR subunits, it is not surprising that several studies, mostly conducted on post-mortem brains, have explored the putative impact of SAP102 and SAP97 alterations on schizophrenia pathophysiology. In a cohort of 34 patients with schizophrenia, SAP97 protein levels were found to be reduced to less than half in the PFC, while SAP102 levels decreased in the hippocampal area compared with HC [142] (Table 2). In the same cohort, the correlation between SAP97 and GluR1 levels in the PFC was found in schizophrenia patients but not in HC, suggesting qualitative differences in the interaction of AMPAR with scaffold proteins [142]. The mRNA expression of SAP102 was reduced in the striatal region of post-mortem brains extracted from patients affected by severe psychiatric diseases, such as schizophrenia, bipolar disorder, and major depression, whereas no changes were observed in the DLPFC and cingulate cortex [133,145]. In another study, SAP102 transcripts levels were decreased in the hippocampus of bipolar disorder I, but not schizophrenia patients, accompanied by a reduction in the expression of NR1 and NR2A mRNA [146]. Moreover, in the PFC of patients affected by bipolar disorder, a decreased SAP102 mRNA expression was detected within small cells of layer II and large cells of layer III [143]. No alterations in SAP-102 protein levels were observed in the dopaminergic neurons of the substantia nigra pars compacta (SNc), as well as in thalamic tissues of subjects with schizophrenia [144,147]. Even preclinical studies explored the relationship between SAP proteins and psychotic disorders. Precisely, in a brain-specific conditional SAP97 KO model, mice exhibited male-Cells 2023, 12, 574 39 of 61 specific cognitive impairments and female-specific motor deficits, along with enrichment for schizophrenia-related genes in the differential gene expression set [97]. Furthermore, haloperidol administration failed to mimic SAP97 changes in rats, suggesting its plausible primary alteration in schizophrenia patients [142]. Overall, these findings highlight the relevance of SAPs proteins in the pathophysiology of psychotic disorders probably through the regulation of glutamatergic receptors at the PSD. Other molecules that play an important role in the structure and function of the PSD are cytoskeletal proteins, such as actin, which interacts with numerous scaffold molecules to spatially organize the PSD [26,36] contributing to a dynamic structure [37]. Indeed, the dendritic spine is constantly changing morphology through the regulation of actin dynamics influenced by the activity of glutamate receptors [373]. Analysis of the spatio-temporal reorganization of postsynaptic substructures during LTP at the level of individual dendritic spines enables distinguishing three sequential phases of protein distribution. In the initial phase, the actin cytoskeleton is readily remodeled while the active cofilin is transported to the spine. Subsequently, a stabilization phase enables the cofilin to form a stable complex with F-actin. The third phase is the remodeling of the PSD by consolidating the position and expansion of the spine [374]. The dynamism of spine morphology is driven by the high concentration of the actin cytoskeleton in spines. Filopodia, which are thin, headless protrusions, are thought to be precursors of dendritic spines, while the drebrin, a protein that binds spines to F-actin, is responsible for the recruitment of F-actin and PSD-95 into filopodia. These components regulate spatially and temporally the morphogenesis of spine [36]. However, the initiation of spine morphogenesis precedes the synaptic assembly of PSD-95 [375] as demonstrated by mutant mice lacking PSD-95 that had normal spine morphology [290]. On the other hand, disruption of the actin cytoskeleton during spine morphogenesis results in the global disassembly of synaptic structural elements [376], suggesting its crucial role in governing spine morphogenesis. The involvement of F-actin in the structural plasticity of mature spines has already been highlighted. However, some authors have hypothesized that actin reorganization could be driven by a local increase in Ca 2+ , triggered by the activation of NMDAR and voltage-gated Ca 2+ channels, providing the molecular basis for activity-dependent actin remodeling [377]. Furthermore, in vitro studies of neuron hippocampal cultures exposed to prolonged NMDAR activity have been shown to induce the disappearance of F-actin at synapses [378]. Moreover, these findings reported that NMDAR-induced loss of actin at spines was time-and concentration-dependent and was blocked by NMDAR antagonists, with a similar effect exerted by L-glutamate, AMPA, and ionomycin, but not by agonists of L-type calcium channels or mGluR. However, the effect of NMDAR activity on local actin assembly was found to be attenuated by pretreatment with an actin-stabilizing compound or an antagonist of the calcium-dependent protein phosphatase calcineurin, suggesting that the actin-mediated stability of the synaptic structure is disturbed by intense glutamate receptor activity and that calcineurin blockers may be useful in preventing such destabilization [378]. It has been hypothesized that in the adult brain actin is responsible for rapid changes in dendritic spines by influencing the function of glutamatergic receptors and, in turn, being influenced by them. Therefore, the actin cytoskeleton is emerging as a crucial mediator in signaling and anatomical plasticity of excitatory synapses [373]. Preclinical studies have suggested a role for actin in experience-modifiable changes in the shape of dendritic spines [379] that have been proposed as the molecular basis for alterations in synaptic connectivity thought to underlie learning and memory [380]. Recent studies have suggested that neuronal encoding of memories may be associated with the dynamic formation and elimination of spines that may be dysfunctional in various pathological conditions [379]. Post-mortem studies have found a reduced density of dendritic spines in the DLPFC of patients with schizophrenia, suggesting that the molecular mechanisms concurring with these alterations are the molecular cascades that contribute to the regulation of the actin cytoskeleton with a potential role in the pathophysiology of schizophrenia [381]. In addition, dysgenesis of dendritic spines is a neuroanatomical finding highly represented in several psychiatric disorders, including intellectual disability, ASD, and schizophrenia [382]. Among the most recently identified synaptic proteins, growing interest has been focused on p140Cap, an adaptor that localizes on dendritic spines contributing to the regulation of synaptogenesis, synaptic transmission, and synaptic plasticity [383][384][385]. Multiple lines of evidence have shown that p140Cap KO mice exhibited cognitive deficits, impaired LTP, LTD, and immature filopodialike dendritic spines. Specifically, p140Cap KO mice showed a defect in LTP and LTD, with reduced object recognition, suggestive of alterations in memory consolidation and cognitive retrieval [385]. A number of p140Cap interacting proteins have been identified in the brain, such as SNAP-25 [384,386], PSD-95 [87], and cortactin, an F-actin-binding protein that regulates actin cytoskeleton organization [387], suggesting that p140Cap acts as a hub for PSD complexes relevant for psychiatric and neurological disorders. Interactors of p140Cap converge on key synaptic processes, including transmission across excitatory synapses, actin cytoskeleton remodeling, and cell-cell junction organization. Remarkably, the interactome of p140Cap and its co-expression network show strong enrichment in genes associated with schizophrenia, ASD, bipolar disorder, intellectual disability, and epilepsy, supporting that synaptic dysfunction is a common biological feature in cerebral diseases [388].

Drebrin in Synaptic Dysfunction and Neuropsychiatric Implications
Dendritic spines receive most of the excitatory synapses in the postsynapse, and their morphological plasticity plays a nodal role in higher brain functions, such as learning and memory. Drebrin is a key modulator of the actin cytoskeleton in neuronal cells, critical for synaptic plasticity, neuritogenesis, and neuronal migration by orchestrating a cross-talk between actin and microtubules. Studies have shown that decreased drebrin levels are a hallmark of multiple neurodegenerative disorders [389]. Loss of drebrin from dendritic spines has been demonstrated to be a pathognomonic feature of synaptic dysfunction associated with neurological disorders accompanied by cognitive deficits [36]. Preclinical in vivo immunoelectron microscopic studies have shown that about 70% of spines contain the PSD protein drebrin under normal conditions that regulate spinogenesis and synaptogenesis. Specifically, drebrin A appears to be implicated in the arrangement of the dendritic actin pool for the composition of spines and excitatory axospinous synapses during the early postnatal periods [390]. However, competitive blockades of NMDAR induce rapid changes in the distribution of F-actin and drebrin A within dendritic spines, suggesting their glutamate-dependent modulation [36,391]. Furthermore, clinical studies have reported that the levels of drebrin A are significantly reduced in the brain of patients with major cognitive impairment, such as Alzheimer's disease [392] and schizophrenia [381]. Interestingly, post-mortem studies have reported decreased drebrin levels in the temporal cortex and increased levels in the frontal cortex of patients with mild cognitive impairment, suggesting a disparity in postsynaptic efficacy in these regions during the onset of Alzheimer's disease. This differential expression of drebrin in the temporal and frontal cortex in Mild Cognitive Impairment patients suggests progressive cognitive dysfunction, associated with initial deterioration of associative behaviors such as memory and language, and relative preservation of executive functions during the early stages of cognitive decline [392].

Spinophilin Regulation in the Dynamic Morphological Changes of Dendritic Spine: Implications in Schizophrenia
Several pieces of evidence have emphasized the role of the PSD protein spinophilin, which is responsible for interacting with several highly enriched proteins in spines [393,394], such as actin [394] and protein phosphatase-1 (PP-1) [394,395]. Several in vitro studies have found that spinophilin is capable of binding actin filaments [393], suggestive of a role as an actin-based cytoskeleton organizer in dendritic spines. Although PP-1 has been shown to modulate the activity of ion channels, including NMDAR [396], this function resulted in the absence of spinophilin. Its role in regulating excitatory glutamatergic transmission by anchoring PP-1 to ionotropic glutamate receptors ( Figure 2). Preclinical studies using spinophilin KO mice have proven that spinophilin modulates both glutamatergic synaptic transmission and dendritic morphology. Specifically, the ability of PP-1 to regulate the activity of NMDARs was found to be reduced in spinophilin KO mice. Additionally, spinophilin gene deletion reported evidence of increased spine density during development and altered filopodial formation in neurons, consistent with its function as a regulator of the dendritic spine [397]. The mechanism by which spinophilin might regulate dynamic morphological changes in the dendritic spine could be an interaction with the actin cytoskeleton [393], which is known to influence spine morphology [398]. In particular, spinophilin can bind F-actin filaments [393] and within the binding domain has several phosphorylation sites for PKA, PKC, and CaMK, which are responsible for regulating cytoskeletal reorganization ( Figure 2). Furthermore, PP-1 has been shown to modulate cell morphology through de-phosphorylation of the actin cytoskeleton [399]. These findings suggest that direct interactions of spinophilin with actin and PP-1 may provide a highly localized mechanism for regulating the phosphorylation state of the actin cytoskeleton. In this context, the modulation of glutamate receptor activity by spinophilin provides another possible effect on spine density as the enhancement of NMDAR transmission by PP-1 inhibitors observed in wild-type mice that was significantly attenuated in KO mice, suggesting that modulation of NMDARs by PP-1 was lost in spinophilin-deficient neurons. Dysregulation of glutamate receptor pathways in spinophilin-deficient neurons appears to lead to specific changes in neuronal circuits [397]. In addition to NMDARs, spinophilin can directly interact with D2Rs implicated in the pathophysiology of schizophrenia and targeted by antipsychotics, suggesting an implication of spinophilin in schizophrenia [394,400] (Figure 3). It has been hypothesized that spinophilin has a role in establishing a signaling complex for dopaminergic neurotransmission through D2Rs, linking receptors to downstream signaling molecules and the actin cytoskeleton [393,400]. Post-mortem studies determined the spinophilin expression in synaptosomal membranes (SPM) and PSD fractions in the DLPFC of schizophrenia patients without antipsychotics vs. antipsychotic-treated and HC. The results of this study showed that spinophilin was significantly reduced in patients with schizophrenia compared with HC in both SPM and PSD fractions (p = 0.007 and p = 0.039, respectively). Moreover, spinophilin was significantly reduced in patients treated with antipsychotics (p = 0.003 for SPM and p = 0.014 for PSD), but not in those without antipsychotics, suggesting that the alterations in the expression of spinophilin potentially might be implicated in the side effects of this class of compound [149].

Discussion and Future Directions
The results reported in this systematic review are discussed here covering three main aspects. Firstly, the main implications that have emerged for PSD proteins with the most robust experimental and clinical data in schizophrenia. Secondly, the role of PSD proteins in the framework of dopamine-glutamate interactions. Thirdly, the translational value of PSD proteins in light of potential innovative therapeutic targets. Multiple lines of evidence support the concept that changes in PSD proteins may be associated with schizophrenia, consistent with the idea of this condition as a synaptopathy [401]. This evidence comes from various "omics" strategies, including GWAS of patients [402], postmortem proteomics and transcriptomics studies [403], as well as in vivo, ex vivo [404], and in vitro [405] models. However, in silico bioinformatics experiments [406] and machine learning applications [407] have contributed in the last year to strengthen the role of PSD proteins in schizophrenia-related synaptopathy [408]. Finally, the possibility that such alterations at the micro-and nano-domain level of the synapse may, acting in synchrony and synergy, extend their consequences to a higher scale of brain function is beginning to be explored by connectivity and connectome analysis [401,409]. Advances in PSDs have unveiled a kind of "molecular Lego" that assembles in the glutamate synapse a sophisticated network in which a single PSD protein (e.g., Shank3 or Homer1) can cooperate on specific signaling or can cooperatively converge by interacting with other PSD proteins on the same signal transduction pathway [8]. This interaction is highly self-organized and occurs rapidly, at least as demonstrated by reconstitution experiments in which PSD proteins exchanged rapidly between condensed fluorescence recovery after photobleaching analyses [410]. The involvement of the Shank protein family, and in particular Shank3, in the genetics of schizophrenia as well as ASD has greatly strengthened the notion that the two classes of disorders, albeit with different ages of onset, clinical trajectory, and diagnostic criteria, may share a genetic background at least for a subgroup of patients. It has confirmed what is already known about the genetics of major psychiatric disorders (bipolar disorder, major depression, and schizophrenia) that share genes particularly involved in immune function, epigenetic regulation, and PSD function. Homer proteins, in particular Homer1 b/c and its early gene isoforms, such as the short Homer1a, act as dominant negatives, have been found to be altered in the post-mortem brain of schizophrenic patients [11] and implicated in modeling the dopamine-glutamate pathophysiology of the preclinical disease proxy [8]. Homer has been shown to play a central role in the construction and modeling (together with PSD-95) of the dendritic spine [50,411,412] and recent evidence has indicated Ankyrin-G as a topologically important crucial node recognized by the EVH1 domain [277]. A more conservative approach to understanding Homer1 in schizophrenia should also take into account the fact that this gene and its transcripts have been associated with broader physiological implications, such as higher cognitive and executive functions, as demonstrated by the effect of genetic variants in modulating working memory [267,268]. Furthermore, DISC1 and Neuregulin are among the PSD proteins with probably the most genetic data available and have been included among the genes contributing to disease susceptibility [10,187]. Considering the peculiar role of these molecules in tuning synaptic function by bridging pre-and postsynaptic structure and function, both molecules extend the role of PSD in disease by highlighting the structural and functional overlapping between pre-and postsynapses [413]. Furthermore, the intersection of Shank3 with the pathophysiology of schizophrenia has recently been marked by the discovery of several point mutations and small deletions of Shank3 [61], as well as the de novo R1117X mutation leading to loss of function of the Shank3 protein causally associated with schizophrenia [64]. Finally, a translational feature directly related to the pharmacotherapy of schizophrenia is the robust effect emerging from animal models of antipsychotic treatment is the differential gene expression at the PSD. This has been demonstrated by antipsychotics alone or in co-administration with other psychotropic drugs, such as mood stabilizers, with and without pretreatment with drugs that mimic the pathophysiology of psychotic behavior [104,116,337]. Critical appraisal of the role of PSD in the pathophysiology of schizophrenia cannot be limited to the evidence and the role, albeit relevant, of proteins that have been firmly implicated in the illness without attempting to place PSD within the overall framework of dopamine-glutamate interaction. In this respect, this interplay appears to be one of the most robust and explored in schizophrenia, although not the only one. It would be conceivable to hypothesize a bidirectional mechanism [414] with dopamine perturbation influencing PSD expression, such as for Homer [415]. Recently, a comprehensive post-mortem analysis was performed in the caudate nucleus of 245 HCs, 154 patients with schizophrenia and 44 patients with bipolar disorder from different countries (210 Africans and 233 Europeans) with an analysis of transancestral quantitative expression loci (eQTLs) and hundreds of specific cis-eQTLs. The integration of quantitative expression loci analysis, Mendelian randomization with the most recent GWAS association study on schizophrenia, the transcriptome-wide association study, and differential expression analysis allowed the identification of many genes associated with schizophrenia risk, potentially including the short isoform of the dopamine D2R. It is plausible to hypothesize that antipsychotic drugs have an influence on caudate gene expression and that their networks highlighted interactions implicated in schizophrenia risk providing a resource for Cells 2023, 12, 574 43 of 61 potential future therapeutic targets [416]. A new and unconventional way of considering the effect of dopamine on the glutamatergic synapse and possibly influencing PSD function derives from the Pajet-Blanc experiment that demonstrated the rapid release of dopamine at striatal varicosities and its effect on the glutamatergic synapse. In this context, the adhesion of dopaminergic synapses to glutamatergic, GABAergic, or cholinergic synapses is organized into a "dopamine hub synapse" supporting local dopaminergic signaling and integrating the volumetric transmission crucial for the modulation of monoamines network activity [417]. Therefore, we could evaluate the current position of PSD in schizophrenia by considering its strengths and weaknesses. Among the strengths, two stand out. The first is the genetic background derived from GWAS or SNPs and the discovery of rare variants that recapitulate several phenotypic aspects of schizophrenia or the syndromic presentation of schizophrenia. The second is the translational value of the preclinical in vivo and ex vivo findings that, by specifying the discrete molecular mechanisms of PSD-mediated synapse function (i.e., growth and formation of dendritic spines), have highlighted the potential molecular mechanisms responsible for the complex behavior and their abnormal expression. Weaknesses include the difficulty in assigning to each protein interacting in a large network (e.g., Homer-PSD95-Shank) with other molecules a specific weight in contributing to a complex disorder such as schizophrenia.
In conclusion, currently, PSD proteins at the glutamatergic synapse level have emerged as one of the major constituents of the dendritic spine implicated in the molecular pathophysiology of schizophrenia, corroborating the concept that this disorder may be inherent at the cellular level as a synaptopathy. Abnormal expression of synaptic function at the micro-macrodomain and higher levels may influence multiple aspects of the pathophysiology of schizophrenia. In this regard, PSD proteins may influence the spatial and temporal organization of dendritic spines, thus are significant players in the complex intersection of neurodevelopment and context-related factors implicated in the pathophysiology of schizophrenia, its clinical phenotypes, and possibly treatment effects.